Immunological studies as well as the analysis of the N termini of different subunits can help establish the actual class of ATPase from A. halophytica. Further characterization of the Na stimulated ATPase in A. halophytica by genetic manipulations is now under way. The purified ATPase from A. halophytica was reconstituted into liposomes to investigate the possible role of Na stimulated ATPase on the transport of Na . The reconstituted ATPase showed catalytic properties, namely Km?s of Na and ATP identical to those of the purified ATPase. Moreover, an increase in the concentration of NaCl and ATP led to an increase in Na uptake by the proteoliposomes . These results suggest the presence of an ATP dependent Na pump in the proteoliposomes. The substrate ATP was hydrolyzed by ATPase to provide a driving force for Na uptake. A previous report demonstrated the function of Na ATPase as a Na pump in the plasma membrane of the marine alga Heterosigma akashiwo based on the similarity of the kinetic properties of Na transport and Na ATPase activity .
In A. halophytica, the apparent Km value of Na transport for Na and ATP were 3.3 and 0.5 mM, respectively. These values are close to the apparent Km values of Na stimulated ATPase activity for Na and ATP . This indicates that the purified ATPase from A. halophytica purchase Iressa is indeed a Na pump. The uptake of Na into proteoliposomes was abolished by gramicidin D and monensin whereas a protonophore CCCP and the permeant anion nitrate had a stimulatory effect on Na uptake . These results suggest the operation of electrogenic Na transport by Na stimulated ATPase in A. halophytica. Moreover, ATP dependent Na uptake by proteoliposomes was a primary and not a secondary event, i.e. the transport was not catalyzed by a Na H antiporter or other secondary events driven by proton potential. As observed in Figure 8A, Na uptake by ATPase is associated with H efflux in A. halophytica.
Previously, the marine alga Tetraselmis viridis was shown to contain Na ATPase capable of translocating Na into plasma membrane vesicles which was accompanied by H efflux with the consequence of the alkalization of the vesicle lumen . In this study, we attempted to elucidate the mechanism of Na and H transport by Na stimulated ATPase reconstituted into liposomes. Two possible mechanisms of Na transport can be hypothesized . Mechanism 1 proposes that the Na stimulated ATPase operates Tyrphostin 9 as a uniporter catalyzing only Na transport across proteoliposomes, subsequently, H transport is driven by the membrane potential generated by Na stimulated ATPase. Mechanism 2 proposes that the Na stimulated ATPase operates as an antiporter catalyzing an exchange of Na for H . To distinguish between these two mechanisms, we studied the role of membrane potential in ATP dependent H translocation.